The ABO blood group system is critically important in blood transfusion. However, ABO incompatibility has had less dramatic impact in hematopoietic transplantation. Since ABO and human leukocyte antigens (HLA) are inherited independently, ABO incompatibility may occur in up to 20-40% of HLA-matched allogeneic hematopoietic stem cell transplants (SCT). Immune hemolysis caused by donor "passenger lymphocytes" contained in the stem cell graft, and pure red cell aplasia (PRCA) associated with prolonged production of recipient type anti-donor isohemagglutinins, have each been described with varying frequency after marrow-derived SCT performed with myeloablative conditioning when there is minor or major ABO incompatibility, respectively, between donor and recipient. However, despite the occurrence of these events, the overall impact of ABO incompatibility in SCT is generally considered low, providing that appropriate transfusion practices are followed. The introduction of PBSC as the hematopoietic graft source, and the use of low intensity nonmyeloablative regimens for recipient conditioning, have dramatically widened the applications of SCT. PBSC are supplanting bone marrow as the preferred stem cell source due to more rapid hematopoietic recovery, improved survival, and relative ease of collection. PBSC also contain an order of magnitude more lymphocytes than bone marrow derived grafts. Low intensity conditioning regimens have further reduced regimen-related toxicity and rely on the generation of graft-versus-tumor immune effects rather than drug or radiation-induced cytotoxic effects to eradicate malignant disease. The combination of PBSC and reduced intensity conditioning are also increasingly being utilized for older patients and for those with debilitating transfusion-dependent non-malignant hematologic diseases. In conjunction with investigators in NHLBI, NCI, and NIAID, the Department of Transfusion Medicine in the Warren Magnuson Clinical Center at NIH has prospectively evaluated the impact of ABO incompatibility following SCT when utilizing PBSC grafts and either conventional myeloablative or reduced intensity non-myeloablative conditioning. We observed massive immune hemolysis in 3 of 10 consecutive patients undergoing HLA-identical, related-donor PBSC transplants with minor ABO incompatibility. Nonablative conditioning was used in 9 of these 10 cases, including two with hemolysis, while cyclosporine alone was used as prophylaxis against graft-versus-host disease (GVHD) in 10/10. Catastrophic hemolysis of 78% of the circulating red cell mass led to anoxic death in the first case, but severe consequences were avoided by early, vigorous donor-compatible red cell transfusions in the subsequent two cases. Hemolysis began 7-11 days after PBSC infusion. A prophylactic 10 unit red cell exchange performed in one patient reduced the circulating recipient-type red cell mass by only 60%, therefore red cell exchanges were not performed in subsequent cases. All patients with hemolysis had a positive direct antiglobulin test (DAT), with eluate reactivity against the relevant recipient antigen. However, neither the intensity of the DAT, the donor isohemagglutinin titer, nor other factors could reliably be used to predict the occurrence of hemolysis. Because immune hemolysis with PBSC grafts has been observed following both ablative and non-ablative conditioning, the increased incidence and severity of hemolysis appears to be most closely related to the PBSC graft lymphocyte content, and use of CsA without an anti-proliferative agent for GVHD prophylaxis. Hemolysis has not been reported using PBSC grafts in which the lymphocyte content is depleted by ex-vivo processing. The DTM continues to evaluate the incidence of hemolysis in this setting, including recent protocols in which mycophenolate mofetil or methotrexate have been added to CsA in the anti-GVHD regimen. To evaluate the effect of major ABO incompatibility on donor red cell engraftment and PRCA following reduced intensity non-myeloablative SCT (NST), we compared consecutive series of patients with major ABO incompatible NST (fludarabine/cyclophosphamide conditioning) and myeloablative SCT (cyclophosphamide/high-dose TBI). Because substantial host hematopoiesis may persist following NST, and hematopoietic and immune function may be both host and donor in origin (mixed chimerism) for prolonged periods, the kinetics of donor erythropoiesis might be expected to differ substantially after major ABO incompatible immune-based NST compared with traditional myeloablative SCT. We found that donor red blood cell (RBC) chimerism (initial detection of donor RBC in peripheral blood) was markedly delayed following NST versus myeloablative SCT, median 114 versus 40 days, and strongly correlated with decreasing host anti-donor isohemagglutinin levels. Anti-donor isohemagglutinins declined to clinically insignificant levels more slowly following NST than myeloablative SCT (median 83 versus 44 days). Donor RBC chimerism was delayed more than 100 days in nine of 14 (64%) and PRCA occurred in four of 14 (29%) patients following NST, while neither event occurred in 12 patients following myeloablative SCT. PRCA lasted 123 to 220 days, and patients with PRCA required a mean of 27 red cell units in the absence of other reasons for transfusion support. Conversion to full donor myeloid chimerism following NST occurred significantly sooner in cases with, compared to those without, PRCA (30 versus 98 days). Patients with a delayed onset of donor red cell chimerism who did not develop PRCA had a delayed conversion to full donor myeloid chimerism, and were protected from reticulocytopenia by a bridge of autologous erythropoiesis. Cyclosporine withdrawal appeared to induce graft-mediated immune effects against recipient isohemagglutinin-producing cells, resulting in decreased anti-donor isohemagglutinin levels and resolution of PRCA following NST. These data indicate that hemolysis may be frequent and severe after transplantation of minor ABO-incompatible PBSCs when utilizing cyclosporine alone to prevent GVHD. We recommend that meticulous clinical monitoring and early, vigorous donor-compatible red cell transfusions should be practiced in all such instances. The data also indicate that significantly delayed donor erythropoiesis may be common following major ABO-incompatible NST and is associated with prolonged persistence of host anti-donor isohemagglutinins. The clinical manifestations of these events are affected by the degree and duration of residual host hematopoiesis, and may be especially significant when utilizing reduced intensity conditioning to treat patients with poor pre-transplant erythropoietic function such as aplastic anemia, sickle cell disease and thalassemia.